CN102875667A - HIV-1 (human immunodeficiency virus-1) peptide Env120-128 specificity TCR (T cell receptor), recombinant retroviral vector thereof and application - Google Patents

Abstract

The invention discloses HIV-1 (human immunodeficiency virus-1) peptide Env120-128 specificity TCR (T cell receptor), a recombinant retroviral vector of HIV-1 peptide Env120-128 specificity TCR and application. According to the HIV-1 peptide Env120-128 specificity TCR and the recombinant retroviral vector, the HIV-1 peptide Env120-128 (KLTPLCVTL) specific TCR gene is successfully sieved, and is subject to trasnfection into the CD8+T cell through a retroviral vector, so that the CD8<+>T cell for expressing the HIV-1 peptide Env120-128 specificity TCR is obtained. The CD8<+>T cell modified by the TCR gene can successfully express the exogenous TCR gene; and the HIV peptide Env120-128 is distinguished based on the specificity, and the IFN-gamma (interferon-gamma) and TNF-alpha (tumor necrosis factor-alpha) cytokines secretion and cytotoxic activity can be mediated; and the application value on treatment of aids and aids/phthisic coinfection disease gene is achieved; and a new way is provided for the adoptive cellular immunotherapy of the aids/phthisic coinfection disease.

Description

A kind of HIV-1 peptide Env 120-128Specificity TCR, its recombinant retroviral vector and application

Technical field

The invention belongs to bioengineering field, be specifically related to a kind of HIV-1 peptide specific φt cell receptor (TCR), and a kind of retroviral vector for AIDS and AIDS/tuberculosis coinfection disease gene treatment that utilizes this TCR to prepare, the CD8 that the retroviral vector transfection obtains ⁺T cell and the application in the anti-AIDS of preparation and AIDS/tuberculosis coinfection disease medicament thereof.

Background technology

Human immunodeficiency virus (Human Immunodeficiency Virus, HIV) be hiv virus, with the havoc body immunity, make various rare infection and cancer be able in human body, occur behind the invasion human body, finally form acquired immune deficiency syndrome (AIDS) (acquired immune deficiency syndrome (AIDS)).HIV mainly is present in HIV the infected or the AIDS patient's body fluid, and any behavior that can cause that body fluid exchanges has the possibility of propagating HIV, comprises transmission through sex, blood propagation and mother-to-baby transmission.Since the eighties in last century, HIV began to propagate in the mankind, nearly 6,000 ten thousand people infected, and wherein 2,500 ten thousand people are dead.

There is no at present the medicine that can eradicate HIV, radical cure acquired immune deficiency syndrome (AIDS), also do not have evidence to show that the people knows from experience HIV generation protective immunity, therefore most of AIDS patients are died from repeatedly secondary infection and tumour.The treatment of acquired immune deficiency syndrome (AIDS) is to suppress virus breeding in vivo on the one hand, strengthens immunologic function; The control opportunistic infection on the other hand, relief of symptoms, extending life.Early treatment and other infection of prevention can delay disease, improve the quality of living.At present methods for the treatment of commonly used has the following aspects clinically: (1) is united " drug cocktail therapy (treatment) " of using several anti-reverse transcription cytotoxic drugs and is implemented antiretroviral therapy, and it has obvious effect in reducing sickness rate and case fatality rate, aspect improving the quality of living; (2) for microbiotic symptomatic treatment and the prevention of various opportunistic infection, comprise chemotherapy and the symptomatic treatment of tumour; (3) other comprise immune modulating treatment, nutritional support treatment and Chinese medicine etc.

HIV-1 peptide Env _120-128It is the peptide epitopes (KLTPLCVTL) of the 120-128 amino acids formation of hiv virus HIV-1 envelope protein gp160, be present in 80% the polytropy HIV-1 peptide sequence, in the strain of HIV-1 B subtype virus, have the conservative property of topnotch, can be expressed the individual recognition of HLA-A*0201.

T cell antigen receptor (T cell receptor, TCR) is the characteristic sign of all T cell surfaces, plays a crucial role in the identification of T cell antigen.TCR is the heterodimer that is made of α, two peptide chains of β, and every peptide chain can be divided into again variable region (V district), constant region (C district), several parts such as cross-film district and cytoplasmic region; Its cytoplasmic region is very short, and the signal transmission is mainly by carrying out with its CD3 molecule of being combined with non covalent bond.TCR molecule contactin, its antigen-specific is present in the V district; Respectively there are again three hypervariable region CDR1, CDR2, CDR3 in V district (V α, V β), and is wherein maximum with the CDR3 variation, directly determined the antigen-binding specificity of TCR.When TCR identification MHC-antigen peptide complex body, CDR1, CDR2 identification and in conjunction with the sidewall of MHC molecular antigen engagement groove, and CDR3 directly combines with antigen peptide.

According to TCR V α, V β gene homology, more than 80 TCR V α genes can be divided into 32 families, more than 60 TCR V β gene is divided into 24 families.Utilize each T cell clone that the characteristics of its unique CDR3 sequence are all arranged, adopt CDR3 spectral pattern analytical technology, can measure the frequency that each CDR3 of each TCR family occurs, reflect thus clone's property of T cell.Do not accept in the T cell of antigenic stimulation, be evenly distributed for the T cell clone of various antigens, show as many families and polyclone, particularly, show as approximately 8 CDR3 peaks that Gaussian distribution all appears being in each family; Antigenic stimulation then causes the some or several specific TCR family t cell responses hyperplasia of this antigen of identification, show as widow clone or mono-clonal distribution that 4 peaks appear being less than in the CDR3 member of this family, the TCR family that wherein has mono-clonal CDR3 distribution (showing as unimodal) namely is the TCR family of antigen-specific mono-clonal hyperplasia.The PCR of this family product is checked order, can obtain antigen-specific TCR CDR3 sequence.

Summary of the invention

The object of the invention is to: filter out HIV-1 peptide Env _120-128(KLTPLCVTL) special tcr gene utilizes retroviral vector to be transfected into CD8 ⁺In the T cell, obtain to express HIV-1 peptide Env _120-128The CD8 of specific TCR ⁺The T cell, and should be through the CD8 of tcr gene modification ⁺The application of T cell in the preparation inverase.

The technical solution adopted in the present invention is:

HIV-1 peptide Env _120-128Specific t-cell receptor (TCR) comprises α chain and β chain, and wherein, the described sequence of SEQ ID NO:3 is contained in the CDR3 district of α chain; The sequence shown in the SEQ ID NO:4 is contained in the CDR3 district of β chain.

Preferably, described HIV-1 peptide Env _120-128The α chain of specificity TCR is to be substituted, to lack and/or increased the aminoacid sequence that one or more amino acid and/or the special and exogenous β chain of end modified rear resulting energy are assembled into the TCR protein molecular by the aminoacid sequence shown in the SEQ ID NO:10; The β chain is to be substituted, to lack and/or increased the aminoacid sequence that one or more amino acid and/or the special and exogenous α chain of end modified rear resulting energy are assembled into the TCR protein molecular by the aminoacid sequence shown in the SEQ ID NO:6.

Preferably, described HIV-1 peptide Env _120-128The α chain amino acid sequence of specificity TCR is shown in SEQ ID NO:12, and the aminoacid sequence of β chain is shown in SEQ ID NO:8.

Coding HIV-1 peptide Env _120-128The gene of specificity TCR.

A kind of HIV-1 peptide Env _120-128The fusion gene of specificity TCR, its sequence is shown in SEQ ID NO:13.

A kind of recombinant retroviral vector contains coding HIV-1 peptide Env _120-128The gene of specificity TCR.

A kind of recombinant retroviral vector contains gene shown in the SEQ ID NO:13.

Preferably, the carrier that sets out of above-mentioned recombinant retroviral vector is pMX-IRES-GFP, pMCs-IRES-GFP or pMYx-IRES-GFP.

The retrovirus that above-mentioned recombinant retroviral vector obtains after packing.

The CD8 of above-mentioned Retroviral Transfer ⁺The T cell.

HIV-1 peptide Env _120-128Specificity TCR, the gene of this TCR that encodes contains recombinant retroviral vector, the retrovirus of this gene, the CD8 of this Retroviral Transfer ⁺The application of T cell in preparation anti-AIDS, AIDS/tuberculosis coinfection disease medicament.

The concrete steps flow process that realizes technique scheme is as follows:

1, screening HIV peptide Env _120-128(KLTPLCVTL) special TCR

1. adopt lymphocyte separation medium to separate HLA-A*0201 type healthy volunteer's peripheral blood mononuclear cell (peripheral blood mononuclear cell, PBMC);

2. count PBMC, adjusting the PBMC number is 1 * 10 ⁶/ hole, every porocyte add respectively and contain HIV peptide Env _120-128The 10% FBS-1640 substratum 2ml of 50ng/ml;

3. after cultivating 2h, add 25U/ml IL-2, added IL-2 to 50U/ml, added IL-2 100U/ml, and continued to cultivate 11 days with this concentration afterwards in the 5th day in the 3rd day;

4. magnetic bead sorting goes out CD8 ⁺The T cell extracts its mRNA, and reverse transcription is cDNA;

5. complementary determining region 3(complementarity determining region 3, and CDR3 spectral pattern before and after CDR3) the spectral pattern analyzing and testing stimulates is found out the HIV peptide Env that is the mono-clonal hyperplasia after the stimulation _120-128(KLTPLCVTL) special TCR α, β gene family.

2, make up recombinant retroviral vector

1. the people TCR α, variable region, β gene family upstream (the variable region that report according to GeneBank, V) and downstream constant region (constant region, C) gene order, design TCR α, β chain full-length gene upstream and downstream primer amplify the special TCR α of HIV peptide, β full-length gene.

2. design contains the restructuring primer of TCR α, β chain downstream, C district and CD3 ζ molecule position of fusion, and the TCR α that the HIV peptide is special, beta gene fragment and CD3 ζ molecule merge.The purpose of this step is to reduce the mispairing of interior exogenous TCR α, β chain, because CD8 ⁺There is the expression of endogenous TCR α, β gene in the T cell, can impel the albumen of exogenous α and beta gene expression correctly to be assembled into TCR protein molecular and stably express at CD8 by replace α, β full-length gene part C district with CD3 ζ chain ⁺The T cell surface can avoid competition in conjunction with CD8 simultaneously ⁺The CD3 molecule of T cell surface, the signal conduction function of exogenous TCR in strengthening improves and modifies rear CD8 ⁺The HIV (human immunodeficiency virus)-resistant activity of T cell.Certainly, can also take other strategies to reduce the mispairing of interior exogenous TCR α, β chain herein, as: with 9 amino acid in α, β chain C district respectively with amino acid substitution (Luo W, Zhang XB, the Huang YT in corresponding site, mouse C district, Hao PP, Jiang ZM, Wen Q, Zhou MQ, Jin Q, Ma L. Development of genetically engineered CD4 ⁺And CD8 ⁺T-cells expressing TCRs specific for a 38 kDa M. tuberculosis antigen. J Mol Med. 2011,89 (9): 903-13); Introduce disulfide linkage (Boulter in exogenous α, β gene C district, J.M. et al. (2003) Stable, soluble T-cell receptor molecules for crystallization and therapeutics. Protein Eng. 16,707-711); Suddenly change exogenous α, β gene C district key amino acid to change the static charge (Voss between α, the β chain, R.H. et al. (2008) Molecular design of the Cab interface favors specific pairing of introduced TCRab in human T cells. J. Immunol. 180,391-401); Exogenous α, β gene V district are merged into a strand TCR and merge (Willemsen with CD3 ζ chain, R.A. et al. (2000) Grafting primary human T lymphocytes with cancer-specific chimeric single chain and two chain TCR. Gene Ther. 7,1369-1377); Utilize 2A to connect exogenous α, β gene realization balance expression (Leisegang M, Engels B, Meyerhuber P, Kieback E, Sommermeyer D, Xue SA, Reuss S, Stauss H, Uckert W. Enhanced functionality of T cell receptor-redirected T cells is defined by the transgene cassette. J Mol Med. 2008,86:573-583.) etc.

3. splice by the α that the HIV peptide is special of the AgeI restriction enzyme site on the F2A-CD3 ζ, β-CD3 ζ fusion gene.

The hV β hC β that 4. will check order correct-CD3 ζ-F2A-hV α hC α-CD3 ζ fusion gene inserts retroviral vector pMX-IRES-GFP and enzyme is cut evaluation.

5. adopt the liposome transfection method, with pMX-hV β hC β-CD3 ζ-F2A-hV α hC α-CD3 ζ-IRES-GFP and envelope protein plasmid VSV-G cotransfection GP2-293 packing cell.

6. collect 48h-72h virus supernatant, the concentrated and purified virus of low temperature ultracentrifugation.

7. recombinant virus infection NIH3T3 cell, the Flow cytometry virus titer, calculation formula: virus titer (IU/ml)=NIH3T3 cell count * GFP positive rate/virus concentrates liquid measure (ml).

3, identify the CD8 of recombinant retrovirus transfection ⁺The tuberculosis of T cell and HIV (human immunodeficiency virus)-resistant activity

1. adopt the Ficoll density gradient centrifugation, separate HLA-A*0201 type donor peripheral blood PBMC;

2. magnetic bead sorting CD8 ⁺The T cell;

3. IL-2 and anti-CD3 monoclonal antibody activate the T cell that sub-elects;

4. by infection multiplicity (multiplicity of infection, MOI)=13 above-mentioned recombinant retrovirus is infected CD8 ⁺The T cell;

5. use IL-2 and anti-CD3 monoclonal antibody to stimulate metainfective CD8 ⁺The T cell;

6. GFP expresses the per-cent of positive cell behind the Flow cytometry virus infection;

7. measure the CD8 of virus transfection ⁺The HIV (human immunodeficiency virus)-resistant activity of T cell:

Experiment arranges: negative control group (HIV peptide Env _120-128The CD8 of specific TCR genetic modification ⁺T cell+dendritic cell (dendritic cells, DC)), untransfected group (untransfected CD8 ⁺T cell+load HIV peptide Env _120-128DC), empty carrier transfection group (empty carrier transfection CD8 ⁺T cell+load HIV peptide Env _120-128DC), irrelevant peptide group (HIV peptide Env _120-128The CD8 of specific TCR genetic modification ⁺T cell+load C MVpp65 _495-503DC), HIVTd+HIV-DC organizes (HIV peptide Env _120-128The CD8 of specific TCR genetic modification ⁺T cell+load HIV peptide Env _120-128DC).

Detect the above-mentioned secretion level of respectively organizing IFN-γ in the cells and supernatant, TNF-α with enzyme linked immunosorbent assay analysis method (enzyme-linked immunosorbent assay, ELISA); Detect CD8 with time resolved fluoro-immunoassay technology (time-resolved fluoroimmuno-assay, TRFIA) ⁺The T cell is to the killing activity of DC.

Wherein, CD8 ⁺The T cell is a kind of cell subsets in the human body, can obtain by separating in the human peripheral, and carry out amplification in vitro and cultivate, and separation and the experimental installation of cultivating require low, technology maturation.

Retroviral vector is the gene delivery vehicle that is made up by a kind of retroviral sequence, can foreign gene-carrying or DNA enter host cell, and be incorporated on the chromogene group, become at present commercially produced product, easily buy and obtain.

The construction process of recombinant retroviral vector is this area molecule clone technology commonly used, the recombinant retrovirus transfection method is animal nutrition commonly used at present, the artificial liposome method of in the present invention, using, can also use other chemical infection protocol, comprise: DEAE-dextran method, calcium phosphate method; And physical method, comprising: microinjection, electroporation, particle gun etc.

Beneficial effect of the present invention is:

The present invention successfully filters out HIV peptide Env _120-128Special TCR carries the CD8 of the Retroviral Transfer of this peptide specific TCR gene ⁺But the exogenous tcr gene of T cell successful expression, specific recognition HIV peptide Env _120-128And mediation IFN-γ, TNF-α cytokine secretion and cytotoxic activity, having the using value of acquired immune deficiency syndrome (AIDS) and AIDS/tuberculosis coinfection disease gene treatment, new footpath is opened up in the cellular immunization treatment of adopting that can be acquired immune deficiency syndrome (AIDS) and AIDS/tuberculosis coinfection disease.

Description of drawings

Fig. 1 HIV peptide Env _120-128CD8 before and after stimulating ⁺T cell TCR α and β chain CDR3 spectral pattern are analyzed;

Fig. 2 recombinant retroviral vector pMX-hV β 18hC β-CD3 ζ-F2A-hV α 11hC α-CD3 ζ-IRES

-GFP makes up schematic diagram;

The enzyme of Fig. 3 pMX-hV β 18hC β-CD3 ζ-F2A-hV α 11hC α-CD3 ζ-IRES-GFP is cut evaluation (M.DL15000 marker; 1. pMX-IRES-GFP empty carrier; 2. the XhoI enzyme of pMX-IRES-GFP empty carrier is cut product; 3. pMX-hV β 18hC β-CD3 ζ-F2A-hV α 11hC α-CD3 ζ-IRES-GFP; 4. the XhoI enzyme of pMX-hV β 18hC β-CD3 ζ-F2A-hV α 11hC α-CD3 ζ-IRES-GFP is cut product; 5. the XhoI+NotI double digestion product of pMX-hV β 18hC β-CD3 ζ-F2A-hV α 11hC α-CD3 ζ-IRES-GFP);

Expression (* 10) (a. light field of NIH3T3 cell GFP after the transfection of Fig. 4 fluorescence microscope recombinant virus; B. fluorescence; C. stacking diagram);

GFP the positive expression rate (a. untransfected of NIH3T3 cell after the transfection of Fig. 5 Flow cytometry recombinant virus; B. pMX-hV β 18hC β-CD3 ζ-F2A-hV α 11hC α-CD3 ζ-IRES-GFP);

CD8 after the transfection of Fig. 6 fluorescence microscope recombinant virus ⁺The expression (* 10 of T cell GFP; EmTd: empty carrier transfection group; HIV Td: the recombinant virus transfection group);

CD8 after the transfection of Fig. 7 Flow cytometry recombinant virus ⁺The expression of T cell GFP (UnTd: untransfected group; EmTd: empty carrier transfection group; HIV Td: the recombinant virus transfection group);

Fig. 8 ELISA detects CD8 ⁺The secretion level of T cell IFN-γ;

Fig. 9 ELISA detects CD8 ⁺The secretion level of T cell TNF-α;

Figure 10 TRFIA detects CD8 ⁺The killing activity of T cell.

Embodiment

The present invention is further illustrated below in conjunction with embodiment, but be not limited to this.

The experimental technique of unreceipted actual conditions in following examples, operate according to normal condition, " molecular cloning experiment guide " (third edition) (the Sambrook J that writes such as Sambrook etc., Russell DW, Janssen K, the yellow training hall of Argentine J. waits translates 2002, Beijing: the condition Science Press), or the condition of advising according to manufacturer.

In following examples, all measurement data results represent with ± s, adopt the relatively difference of cytokine IFN-γ, TNF-α secretion level between each group of one-way analysis of variance (One-Way ANOVA), proofread and correct with Welch during heterogeneity of variance, adopt the LSD method to carry out comparing in twos between each group, adopt Dunnett ' s T3 method to proofread and correct during heterogeneity of variance.Inspection level α=0.05, two-tailed test.Adopt SPSS17.0 for windows statistical package to carry out data analysis.

Embodiment

1. screen HIV-1 peptide Env _120-128 (KLTPLCVTL) special TCR

1.1 density gradient centrifugation separation and purification PBMC

(1) adds an amount of Ficoll lymphocyte separation medium at the aseptic centrifuge tube of 15ml scale;

(2) the abundant mixing dilution of the peripheric venous blood of taking heparin anti-freezing and equivalent RPMI 1640 liquid, the anticoagulation of drawing 2 times of volumes with the pasteur dropper slowly is superimposed on the lymphocyte separation medium along tube wall, notes keeping the interface complete.18 ~ 20 ° of C, 1800 ~ 2000rpm/min horizontal centrifugal, 20 ~ 30min;

(3) centrifugal rear liquid in pipe is divided into four layers, and the upper strata is blood plasma and diluent, and the pipe end is mainly red corpuscle and GCL.The middle level is lymphocyte separation medium, has at the interface one in upper, middle level take mononuclearcell as main canescence cloud and mist layer;

(4) be inserted into buffy coat with suction pipe, draw mononuclearcell, place in another centrifuge tube, add 5 times with RPMI 1640 liquid of upper volume, 18 ~ 20 ° of C, the centrifugal 10min of 1500rpm/min is PBMC behind the thrombocyte that twice removal of washed cell major part mixes;

(5) cell counting and cell viability detect: the PBMC cell suspension mixes with the blue dye liquor of 0.4 % platform phenol of 1/10 volume, four total cell count of large grid on angle on the tally on the blood counting chamber, and 1/4th numbers of total cell count multiply by 10 ⁴Be every ml concn; Dead cell pigmentable trypan blue, that lives is not painted, counts 200 lymphocytes, calculating viable cell percentage living cell rate %=(viable count/total cell count) * 100%.

1.2 preparation HIV peptide Env _120-128Special T cell clone:

(1) counting PBMC, adjusting the PBMC number is 1 * 10 ⁶/ hole adds respectively and contains HIV-1 peptide Env _120-128The 10% FBS-1640 substratum 2ml of 50ng/ml;

(2) 37 ° of C, 5% CO ₂After cultivating 2h under the condition, add IL-2 25U/ml;

Added IL-2 to 50U/ml in (3) the 3rd days, added IL-2 100U/ml, and continued to cultivate 11 days with this concentration afterwards in the 5th day.

1.3 immunomagnetic beads (beautiful day Ni biotech firm of Germany) sorting CD8 ⁺The T cell:

1.4 total RNA extraction reagent box (OMEGA) extracts total RNA of the above-mentioned cell precipitation of collecting:

1.5 reverse transcription (RT) test kit (Fermentas) synthesizes cDNA.

1.6 34 TCR V of pcr amplification α gene family CDR3 fragment (reference literature: XIN-SHENG etc., 2006, Clinical ﹠amp; Laboratory Haematology, 28:405-415.):

Utilize that 34 TCR V α family specificity upstream primers and shared downstream C α are outer, inboard primer is done heminested PCR:

First round PCR: every sample is done 34 PCR reaction tubess, and the 1st ~ 34 pipe adds respectively TCR V α 1 to V α 34 family's upstream primers, and every pipe adds shared downstream C α outside primer 1 μ l, and each primer concentration is 10 μ M.Every PCR reaction tubes volume is 25 μ l, contains cDNA template 1.0 μ l, 10mmol/L dNTP 0.5 μ l, 10 * Buffer, 2.5 μ l, 25mmol/LMgCl ₂1.5 μ l, Taq archaeal dna polymerase 0.625U.PCR reaction conditions: 95 ° of C denaturation 3min; 95 ° of C 30s, 60 ° of C 30s, 72 ° of C 1min, 35 circulations; 72 ° of C extend 10min.

Second takes turns PCR: the reaction cumulative volume is 25 μ l, contains first round PCR product 2 μ l, 10mmol/L dNTP 0.5 μ l, 10 * Buffer, 2.5 μ l, 25mmol/L MgCl ₂1.5 μ l, Taq archaeal dna polymerase 0.625U, 34 family's upstream primers of TCR V α, 1 μ l, the inboard C α of downstream FAM mark primer 1 μ l, each primer concentration is 10 μ M.PCR reaction conditions: 95 ° of C 2min; 60 ° of C 2min, 72 ° of C 10min, 4 circulations.

1.7 24 TCR V of pcr amplification β gene family CDR3 fragment (reference literature: XIN-SHENG etc., 2006, Clinical ﹠amp; Laboratory Haematology, 28:405 – 415.):

Every sample is done 24 PCR reaction tubess, and every pipe adds TCR C β-FAM downstream primer 1 μ l, and the 1st to the 24th pipe adds respectively TCR V β 1 to TCR V β 24 upstream primers 1 μ l, and each primer concentration is 10 μ M.The PCR reaction volume is 25 μ l, contains cDNA template 1 μ l, 10mmol/L dNTP 0.5 μ l, 10 * Buffer, 2.5 μ l, 25mmol/L MgCl ₂1.5 μ l, Taq archaeal dna polymerase 0.625U.PCR reaction conditions: 94 ° of C sex change 3min; 94 ° of C 1min, 55 ° of C 1min, 72 ° of C 1min, 35 circulations; 72 ° of C extend 10min.

1.8 agarose gel electrophoresis:

Get 34 TCR V α and 24 each 5 μ l of TCR V β gene family PCR product, 2% agarose gel electrophoresis, 110V, 20min adopts gel imaging system to take a picture.Residue PCR product-20 a ° C saves backup.

1.9 the CDR3 spectral pattern is analyzed:

Get 34 V α, 24 V β gene family FAM fluorescent mark PCR product 2 μ l, at 373DNA sequenator (ABI, Perkin Elmer) carries out 6% polyacrylamide gel electrophoresis on, collect the fluorescent signal of the varying strength that different time occurs in the electrophoresis process, the data that GeneScan 672 software automatic analysis are collected, be converted to the peak of different positions, height and form, represent the frequency that each CDR3 member of TCR family occurs, reflect thus clone's property of each TCR family.Wherein, the TCR family that has a unimodal distribution namely is the TCR family of antigen-specific mono-clonal hyperplasia.

CDR3 spectral pattern analytical results shows, antigen peptide stimulation CD8 ⁺Behind the T cell, part tcr gene family spectral pattern changes, and by original 8 or become the few peak of the list that is less than 8 peaks more than the Gaussian distribution of 8 peak types and distribute, shows that these families are because antigen peptide continues to stimulate widow clone or the mono-clonal hyperplasia that causes.The variation of CDR3 spectral pattern before and after the comparison stimulus, finding out stimulates the front polyclone that is, is respectively V α 11, V β 18 gene families (Fig. 1) of mono-clonal amplification after the HIV peptide stimulates.

Order-checking shows, HIV-1 peptide Env _120-128(KLTPLCVTL) nucleotide sequence in the CDR3 district of special TCR V α 11, V β 18 is shown in SEQ ID NO:1 and SEQ ID NO:2, and the aminoacid sequence of two CDR3 sequence encodings is respectively shown in SEQ ID NO:3 and SEQ ID NO:4.

2. make up recombinant retroviral vector (make up flow process and see Fig. 2)

2.1 synthetic primer

According to TCR CDR3 spectral pattern, filter out and be polyclone peak type before the stimulation, be unimodal TCR V α, V β gene family after stimulating, this gene family V region sequence design full-length gene upstream and downstream primer according to the GeneBank report, according to the restructuring primer of bibliographical information design with fusion place of CD3 ζ molecule, according to F2A peptide catenation sequence design restructuring primer, all primer is synthetic by Invitrogen Shanghai Ying Jun Bioisystech Co., Ltd, and (5 ' to 3 ') is as follows for primer title and sequence:

2.2 pcr amplification hV β 18hC β-CD3 ζ-F2A-hV α 11hC α-CD3 ζ fusion gene:

(1) take the cDNA of step 1.5 preparation as template, utilize primer P1 and P2, the Pfu archaeal dna polymerase, in the pcr amplification to hC β 393bp place fragment and catenation sequence ggggat and 5 ' the sequence S1(hV β 18hC β district of holding CD3 ζ).HV β 18hC β full-length gene order is shown in SEQ ID NO:5, and its coded aminoacid sequence is shown in SEQ ID NO:6.

(2) take the cDNA of step 1.5 preparation as template, utilize primer P3 and P4, Pfu archaeal dna polymerase, pcr amplification hC β 393bp fore portion sequence, catenation sequence ggggat, CD3 ζ full length sequence and 5 ' end F2A sequence S2(hC β _393bp-CD3 ζ-5 ' end F2A district).

(3) S2 that the S1 that obtains with step (1) and step (2) obtain utilizes primer P1 and P4 as template, Pfu archaeal dna polymerase, recombinant PCR amplification hV β 18hC β _393bp-CD3 ζ-5 ' end F2A sequence S3(is hV β 18hC β wherein _393bpThe sequence of-CD3 ζ section is shown in SEQ ID NO:7, and its coded aminoacid sequence is shown in SEQ ID NO:8).

(4) take the cDNA of step 1.5 preparation as template, utilize primer P5 and P6, the Pfu archaeal dna polymerase, sequence S4(3 ' to hC α 284bp place's fragment and catenation sequence ggggat and 5 ' end CD3 ζ among pcr amplification 3 ' the end F2A-hV α 11hC α (wherein the nucleotide sequence of hV α 11hC α is shown in SEQ ID NO:9, and its coded aminoacid sequence is shown in SEQ ID NO:10) holds F2A-hV α 911hC α district).

(5) take the cDNA of step 1.5 preparation as template, utilize primer P7 and P8, Pfu archaeal dna polymerase, pcr amplification hC α 284bp fore portion sequence, catenation sequence ggggat and CD3 ζ full length sequence S5(hC α _284bp-CD3 ζ district).

(6) S5 that the S4 that obtains with step (4) and step (5) obtain utilizes primer P5 and P8 as template, Pfu archaeal dna polymerase, recombinant PCR amplification 3 ' end F2A-hV α 11hC α _284bp-CD3 ζ sequence S6.Wherein, hV α 11hC α _284bpThe sequence of-CD3 ζ section is shown in SEQ ID NO:11, and its coded aminoacid sequence is shown in SEQ ID NO:12.

(7) utilize restriction enzyme A geI single endonuclease digestion S3 and S6, gel reclaims the fragment after test kit (Omega) Separation and Recovery enzyme is cut and connects to obtain hV β 18hC β-CD3 ζ-F2A-hV α 11hC α-CD3 ζ (SEQ ID NO:13) gene fragment with the T4 dna ligase.

Above conventional PCR reaction system 25 μ l contain 10 * Buffer, 2.5 μ l, 10mmol/L dNTP 0.5 μ l, Pfu archaeal dna polymerase 0.2U, each 0.8 μ l of 10 μ M primers, template DNA 0.5 μ l.Conventional PCR reaction conditions: 95 ° of C sex change 5min; 95 ° of C 1min, 63 ° of C 1min, 72 ° of C 1min, 35 circulations; 72 ° of C extend 10min.

Recombinant PCR reaction system 25 μ l contain 10 * Buffer, 2.5 μ l, 10mmol/L dNTP 0.8 μ l, Pfu archaeal dna polymerase 0.2U, each 0.6 μ l of 10 μ M primers, two kinds of each 6 μ l of PCR product template.PCR reaction conditions: 95 ° of C sex change 5min; 95 ° of C 1min, 50 ° of C 30s, 72 ° of C 1min, 3 circulations; 95 ° of C 90s, 65 ° of C 30s, 72 ° of C 90s, 32 circulations; 72 ° of C extend 10min.

2.3 make up the cloning vector that contains hV β 18hC β-CD3 ζ-F2A-hV α 11hC α-CD3 ζ gene fragment

(1) reclaims test kit (Omega) with glue and reclaim hV β 18hC β-CD3 ζ-F2A-hV α 11hC α-CD3 ζ gene fragment;

(2) add A with DNA A-Tailing test kit (TaKaRa) at said gene fragment end;

(3) with in hV β 18hC β-CD3 ζ-F2A-hV α 11hC α-CD3 ζ gene fragment access pGEM-T carrier, ligation system 10 μ l:pGEM-T carriers 1 μ l, 10 * ligation Buffer, 1 μ l, T4 dna ligase 1 μ l, 0.2pmol have added the PCR product of A purifying, and 16 ° of C connections are spent the night.

(4) ordinary method will connect correct Plasmid Transformation E.coli DH5 α competence bacteria.Then bacterium is coated on the penbritin flat board of 4 μ l 200mg/ml IPTG, 40 μ l, 20 mg/ml X-gal overnight incubation.The bacterium colony of recombinant plasmid transformed is and is white, and the bacterium colony that empty plasmid transforms is blue.Select dull and stereotyped upper white colony, forward to 3ml Amp is housed ⁺In the test tube of LB nutrient solution, 37 ° of C, 160rpm jolting 12-16h.

(5) extract plasmid with plasmid extraction test kit (Omega), with corresponding restriction enzyme the positive recombinant plasmid of primary dcreening operation is identified, and take recombinant plasmid as template, carried out pcr amplification, agarose gel electrophoresis is identified clip size.Send Invitrogen Shanghai Ying Jun Bioisystech Co., Ltd to check order the primary dcreening operation positive plasmid at last.Sequencing result shows, exogenous gene sequence and forecasting sequence that this recombinant plasmid contains are in full accord.

2.4 the structure of recombinant retroviral vector

(1) cut T carrier and the pMX-IRES-GFP empty plasmid that contains hV β 18hC β-CD3 ζ-F2A-hV α 11hC α-CD3 ζ fusion gene with restriction enzyme Xho I, Not I enzyme, glue reclaims hV β 18hC β-CD3 ζ-F2A-hV α 11hC α-CD3 ζ fusion gene fragment and vector gene fragment (method is with step 2.3) respectively;

(2) after enzyme is cut after hV β 18hC β-CD3 ζ-F2A-hV α 11hC α-CD3 ζ fusion gene fragment connects into pMX-IRES-GFP carrier (method is with step 2.3), transformed competence colibacillus bacterium XL-10, after coating the solid medium cultivation 12-16h that contains penbritin, select single bacterium colony, shake bacterium and spend the night;

(3) extracting plasmid, enzyme are cut qualification result and are shown, gene fragment is inserted correct (Fig. 3);

(4) select positive bacteria to drop into capable amplification cultivation, a large amount of extracting plasmid DNA.

2.5 the packing of retrovirus recombinant vectors

Recombinant plasmid mixes with the ratio of 1:1 with envelope protein plasmid VSV-G, adopts liposome transfection method transfection GP2-293 cell, packing retrovirus, the Lipofectamine 2000 test kit description operation that Invitrogen is pressed in experiment.

2.6 recombinant retrovirus is concentrated and purified

(1) collects viral supernatant, 10000g, 4 ° of centrifugal 10min of C;

(2) reclaim viral supernatant, 50000g, 4 ° of centrifugal 2h of C;

(3) TNE of 1%-3% original volume is resuspended, after virus is dissolved fully, and packing ,-80 ° of C store;

2.7 Flow Cytometry Assay virus titer

(1) in advance with NIH3T3 cell (2 * 10 ⁵/ hole) inoculation culture 24h;

(2) add polybrene (PB) to final concentration 8mg/L, add the viral supernatant of 10 μ l titre to be measured;

(3) behind the infection 24h, change fresh medium, remove pseudovirion;

(4) 37 ° of C observe the expression of green fluorescent protein (GFP) after continuing to cultivate 3d under the inverted fluorescence microscope;

After (5) 37 ° of C continued to cultivate 5d, after trysinization, PBS washed 3 times, resuspended with 200-300 μ l PBS, preparation density was 1-5 * 10 ⁶The single cell suspension of/ml, flow cytometer detect its GFP the positive expression rate, calculate virus titer by following formula: virus titer (GFU/ml)=NIH3T3 cell count * positive rate/transfection virus supernatant amount (ml).

The fluorescence microscopy Microscopic observation, the NIH3T3 cell expressing green fluorescence of the retroviral infection of packing through the GP2-293 cell shows the expression (Fig. 4) of GFP in cell.The titre that calculates recombinant virus is 1.59 * 10 ⁷IU/mL(Fig. 5).

3. recombinant retrovirus infects CD8 ⁺ The T cell

3.1 with CD8 ⁺T cell infection the day before yesterday is with 5 * 10 ⁵Individual/hole is inoculated in 24 orifice plates;

3.2 the same day was abandoned the old liquid of cell cultures in transfection, was the viral stock solutions of 13 addings by MOI, adding PB is 8mg/L to final concentration, and 37 ° of C cultivate 4h;

3.3 the adding substratum, dilution PB to 2mg/L continues to cultivate 5 days;

3.4 centrifugal collecting cell, PBS washing 2 times, 2% Paraformaldehyde 96 is fixed;

3.5 flow cytometry analysis CD8 ⁺T cell GFP the positive expression rate is 29.5%(Fig. 6,7).

4. identify the CD8 of recombinant retrovirus transfection ⁺ The HIV (human immunodeficiency virus)-resistant activity of T cell

4.1 ELISA test kit (Wuhan Boster Biological Technology Co., Ltd.) is measured CD8 ⁺The IFN-γ of T cell, TNF-α secretion level

The experiment contrast group arranges: negative control group (HIVTd+DC:HIV peptide Env _120-128The CD8 of specific TCR genetic modification ⁺T cell+DC), untransfected group (UnTd+DC-HIV: untransfected CD8 ⁺T cell+load HIV peptide Env _120-128DC), empty carrier transfection group (EmTd+DC-HIV: empty carrier transfection CD8 ⁺T cell+load HIV peptide Env _120-128DC), irrelevant peptide group (HIVTd+DC-CMV:HIV peptide Env _120-128The CD8 of specific TCR genetic modification ⁺The DC of T cell+load C MVpp65), HIVTd+HIV-DC group (HIV peptide Env _120-128The CD8 of specific TCR genetic modification ⁺T cell+load HIV peptide Env _120-128DC).Following experiment contrast arranges all herewith.Experiment repeats 3 times, and method is as follows:

(1) DC is with 5 * 10 ³Individual/hole is inoculated in 96 orifice plates, E:T=7 when surveying the secretory volume of IFN-γ by the E:T(that groped, E:T=20 when surveying the secretory volume of TNF-α) add CD8 ⁺The T cell is established three multiple holes for every group;

(2) CD8 ⁺T cell and DC mixed culture certain hour (mixed culture 18h when surveying IFN-γ, mixed culture 24h when surveying TNF-α) are collected each hole culture supernatant, operate by the test kit specification sheets.

ELISA result shows:

1. imitating target than (E:T)=7, with load HIV peptide Env _120-128DC mixed culture 18h the time, the CD8 of HIV peptide specific TCR genetic modification ⁺T cell IFN-γ secretion level reaches maximum 1401.667 ± 81.969pg/ml, the CD8 that be significantly higher than untransfected, dallies and dye ⁺T cell and the CD8 that modifies with the tcr gene that the DC of load HIV peptide or other irrelevant peptide of load not cultivates altogether ⁺The T cell ( P＜0.001), sees Fig. 8;

2. at E:T=20, with load HIV peptide Env _120-128DC mixed culture 24h the time, the CD8 of HIV peptide specific TCR genetic modification ⁺T cell TNF-α secretion level reaches maximum 1009.22 ± 67.073pg/ml, the CD8 that be significantly higher than untransfected, dallies and dye ⁺T cell and the CD8 that modifies with the tcr gene that the DC of load HIV peptide or other irrelevant peptide of load not cultivates altogether ⁺The T cell ( P＜0.001), sees Fig. 9.

4.2 time resolved fluoro-immunoassay test kit (PerkinElmer) is measured CD8 ⁺The T cell killing activity operates by the test kit specification sheets.

Temporal resolution immunofluorescence detected result shows: at E:T=30, with load HIV peptide Env _120-128DC mixed culture 4h the time, the CD8 that tcr gene is modified ⁺The T cell killing activity is the highest, reaches 46.45%, the CD8 that be significantly higher than untransfected, dallies and dye ⁺T cell and the CD8 that modifies with the tcr gene that the DC of load HIV peptide or other irrelevant peptide of load not cultivates altogether ⁺The level of killing and wounding of T cell ( P＜0.001), sees Figure 10.

Above-mentioned experimental result shows: carry HIV peptide Env _120-128The CD8 of the Retroviral Transfer of specific TCR gene ⁺But the exogenous tcr gene of T cell successful expression, specific recognition HIV peptide Env _120-128And mediation IFN-γ, TNF-α cytokine secretion and cytotoxic activity, the using value that has acquired immune deficiency syndrome (AIDS) and treat with tuberculosis coinfection disease gene can be acquired immune deficiency syndrome (AIDS) and new footpath is opened up in the cellular immunization treatment of adopting lungy.

＜110〉Nanfang Medical Univ

＜120〉a kind of HIV-1 peptide Env _120-128Specificity TCR, its recombinant retroviral vector and application

<130>

<160> 21

<170> PatentIn version 3.5

<210> 1

<211> 63

<212> DNA

<213> human

<400> 1

gcctctgggg gttaccagaa agttaccttt ggaactggaa caaagctcca agtcatccca 60

aat 63

<210> 2

<211> 57

<212> DNA

<213> human

<400> 2

gcccgggacg gttcctacga gcagtacttc gggccgggca ccaggctcac ggtcaca 57

<210> 3

<211> 21

<212> PRT

<213> human

<400> 3

Ala Ser Gly Gly Tyr Gln Lys Val Thr Phe Gly Thr Gly Thr Lys Leu

1 5 10 15

Gln Val Ile Pro Asn

20

<210> 4

<211> 19

<212> PRT

<213> human

<400> 4

Ala Arg Asp Gly Ser Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu

1 5 10 15

Thr Val Thr

<210> 5

<211> 942

<212> DNA

<213> human

<400> 5

atggacacca gagtactttg ctgtgcggtc atctgtcttc tgggggcagg tctctcaaat 60

gccggcgtca tgcagaaccc aagacacctg gtcaggagga ggggacagga ggcaagactg 120

agatgcagcc caatgaaagg acacagtcat gtttactggt atcggcagct cccagaggaa 180

ggtctgaaat tcatggttta tctccagaaa gaaaatatca tagatgagtc aggaatgcca 240

aaggaacgat tttctgctga atttcccaaa gagggcccca gcatcctgag gatccagcag 300

gtagtgcgag gagattcggc agcttatttc tgtgccagct cacgcgcccg ggacggttcc 360

tacgagcagt acttcgggcc gggcaccagg ctcacggtca cagaggacct gaaaaacgtg 420

ttcccacccg aggtcgctgt gtttgagcca tcagaagcag agatctccca cacccaaaag 480

gccacactgg tgtgcctggc cacaggcttc taccccgacc acgtggagct gagctggtgg 540

gtgaatggga aggaggtgca cagtggggtc agcacagacc cgcagcccct caaggagcag 600

cccgccctca atgactccag atactgcctg agcagccgcc tgagggtctc ggccaccttc 660

tggcagaacc cccgcaacca cttccgctgt caagtccagt tctacgggct ctcggagaat 720

gacgagtgga cccaggatag ggccaaacct gtcacccaga tcgtcagcgc cgaggcctgg 780

ggtagagcag actgtggctt cacctccgag tcttaccagc aaggggtcct gtctgccacc 840

atcctctatg agatcttgct agggaaggcc accttgtatg ccgtgctggt cagtgccctc 900

gtgctgatgg ccatggtcaa gagaaaggat tccagaggct ag 942

<210> 6

<211> 312

<212> PRT

<213> human

<400> 6

Asp Thr Arg Val Leu Cys Cys Ala Val Ile Cys Leu Leu Gly Ala Gly

1 5 10 15

Leu Ser Asn Ala Gly Val Met Gln Asn Pro Arg His Leu Val Arg Arg

20 25 30

Arg Gly Gln Glu Ala Arg Leu Arg Cys Ser Pro Met Lys Gly His Ser

35 40 45

His Val Tyr Trp Tyr Arg Gln Leu Pro Glu Glu Gly Leu Lys Phe Met

50 55 60

Val Tyr Leu Gln Lys Glu Asn Ile Ile Asp Glu Ser Gly Met Pro Lys

65 70 75 80

Glu Arg Phe Ser Ala Glu Phe Pro Lys Glu Gly Pro Ser Ile Leu Arg

85 90 95

Ile Gln Gln Val Val Arg Gly Asp Ser Ala Ala Tyr Phe Cys Ala Ser

100 105 110

Ser Arg Ala Arg Asp Gly Ser Tyr Glu Gln Tyr Phe Gly Pro Gly Thr

115 120 125

Arg Leu Thr Val Thr Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val

130 135 140

Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala

145 150 155 160

Thr Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu

165 170 175

Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp

180 185 190

Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys

195 200 205

Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg

210 215 220

Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp

225 230 235 240

Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala

245 250 255

Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln

260 265 270

Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys

275 280 285

Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met

290 295 300

Val Lys Arg Lys Asp Ser Arg Gly

305 310

<210> 7

<211> 1224

<212> DNA

＜213〉artificial sequence

<400> 7

atggacacca gagtactttg ctgtgcggtc atctgtcttc tgggggcagg tctctcaaat 60

gccggcgtca tgcagaaccc aagacacctg gtcaggagga ggggacagga ggcaagactg 120

agatgcagcc caatgaaagg acacagtcat gtttactggt atcggcagct cccagaggaa 180

ggtctgaaat tcatggttta tctccagaaa gaaaatatca tagatgagtc aggaatgcca 240

aaggaacgat tttctgctga atttcccaaa gagggcccca gcatcctgag gatccagcag 300

gtagtgcgag gagattcggc agcttatttc tgtgccagct cacgcgcccg ggacggttcc 360

tacgagcagt acttcgggcc gggcaccagg ctcacggtca cagaggacct gaaaaacgtg 420

ttcccacccg aggtcgctgt gtttgagcca tcagaagcag agatctccca cacccaaaag 480

gccacactgg tgtgcctggc cacaggcttc taccccgacc acgtggagct gagctggtgg 540

gtgaatggga aggaggtgca cagtggggtc agcacagacc cgcagcccct caaggagcag 600

cccgccctca atgactccag atactgcctg agcagccgcc tgagggtctc ggccaccttc 660

tggcagaacc cccgcaacca cttccgctgt caagtccagt tctacgggct ctcggagaat 720

gacgagtgga cccaggatag ggccaaacct gtcacccaga tcgtcagcgc cgaggcctgg 780

ggtagagcag actgtgggga tctggatccc aaactctgct acctgctgga tggaatcctc 840

ttcatctatg gtgtcattct cactgccttg ttcctgagag tgaagttcag caggagcgca 900

gacgcccccg cgtaccagca gggccagaac cagctctata acgagctcaa tctaggacga 960

agagaggagt acgatgtttt ggacaagaga cgtggccggg accctgagat ggggggaaag 1020

ccgcagagaa ggaagaaccc tcaggaaggc ctgtacaatg aactgcagaa agataagatg 1080

gcggaggcct acagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa ggggcacgat 1140

ggcctttacc agggtctcag tacagccacc aaggacacct acgacgccct tcacatgcag 1200

gccctacccc ctcgcggctc cgga 1224

<210> 8

<211> 430

<212> PRT

＜213〉artificial sequence

<400> 8

Asp Thr Arg Val Leu Cys Cys Ala Val Ile Cys Leu Leu Gly Ala Gly

1 5 10 15

Leu Ser Asn Ala Gly Val Met Gln Asn Pro Arg His Leu Val Arg Arg

20 25 30

Arg Gly Gln Glu Ala Arg Leu Arg Cys Ser Pro Met Lys Gly His Ser

35 40 45

His Val Tyr Trp Tyr Arg Gln Leu Pro Glu Glu Gly Leu Lys Phe Met

50 55 60

Val Tyr Leu Gln Lys Glu Asn Ile Ile Asp Glu Ser Gly Met Pro Lys

65 70 75 80

Glu Arg Phe Ser Ala Glu Phe Pro Lys Glu Gly Pro Ser Ile Leu Arg

85 90 95

Ile Gln Gln Val Val Arg Gly Asp Ser Ala Ala Tyr Phe Cys Ala Ser

100 105 110

Ser Arg Ala Arg Asp Gly Ser Tyr Glu Gln Tyr Phe Gly Pro Gly Thr

115 120 125

Arg Leu Thr Val Thr Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val

130 135 140

Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala

145 150 155 160

Thr Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu

165 170 175

Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp

180 185 190

Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys

195 200 205

Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg

210 215 220

Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp

225 230 235 240

Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala

245 250 255

Glu Ala Trp Gly Arg Ala Asp Cys Gly Asp Leu Asp Pro Lys Leu Cys

260 265 270

Tyr Leu Leu Asp Gly Ile Leu Phe Ile Tyr Gly Val Ile Leu Thr Ala

275 280 285

Leu Phe Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr

290 295 300

Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg

305 310 315 320

Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met

325 330 335

Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn

340 345 350

Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met

355 360 365

Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly

370 375 380

Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala

385 390 395 400

Leu Pro Pro Arg Gly Ser Gly Ala Pro Val Lys Gln Thr Leu Asn Phe

405 410 415

Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro Gly

420 425 430

<210> 9

<211> 813

<212> DNA

<213> human

<400> 9

atgaagccca ccctcatctc agtgcttgtg ataatattta tactcagagg aacaagagcc 60

cagagagtga ctcagcccga gaagctcctc tctgtcttta aaggggcccc agtggagctg 120

aagtgcaact attcctattc tgggagtcct gaactcttct ggtatgtcca gtactccaga 180

caacgcctcc agttactctt gagacacatc tctagagaga gcatcaaagg cttcactgct 240

gaccttaaca aaggcgagac atctttccac ctgaagaaac catttgctca agaggaagac 300

tcagccatgt attactgtgc tctaagtgcc tctgggggtt accagaaagt tacctttgga 360

actggaacaa agctccaagt catcccaaat atccagaacc ctgaccctgc cgtgtaccag 420

ctgagagact ctaaatccag tgacaagtct gtctgcctat tcaccgattt tgattctcaa 480

acaaatgtgt cacaaagtaa ggattctgat gtgtatatca cagacaaaac tgtgctagac 540

atgaggtcta tggacttcaa gagcaacagt gctgtggcct ggagcaacaa atctgacttt 600

gcatgtgcaa acgccttcaa caacagcatt attccagaag acaccttctt ccccagccca 660

gaaagttcct gtgatgtcaa gctggtcgag aaaagctttg aaacagatac gaacctaaac 720

tttcaaaacc tgtcagtgat tgggttccga atcctcctcc tgaaagtggc cgggtttaat 780

ctgctcatga cgctgcggct gtggtccagc tga 813

<210> 10

<211> 269

<212> PRT

<213> human

<400> 10

Lys Pro Thr Leu Ile Ser Val Leu Val Ile Ile Phe Ile Leu Arg Gly

1 5 10 15

Thr Arg Ala Gln Arg Val Thr Gln Pro Glu Lys Leu Leu Ser Val Phe

20 25 30

Lys Gly Ala Pro Val Glu Leu Lys Cys Asn Tyr Ser Tyr Ser Gly Ser

35 40 45

Pro Glu Leu Phe Trp Tyr Val Gln Tyr Ser Arg Gln Arg Leu Gln Leu

50 55 60

Leu Leu Arg His Ile Ser Arg Glu Ser Ile Lys Gly Phe Thr Ala Asp

65 70 75 80

Leu Asn Lys Gly Glu Thr Ser Phe His Leu Lys Lys Pro Phe Ala Gln

85 90 95

Glu Glu Asp Ser Ala Met Tyr Tyr Cys Ala Leu Ser Ala Ser Gly Gly

100 105 110

Tyr Gln Lys Val Thr Phe Gly Thr Gly Thr Lys Leu Gln Val Ile Pro

115 120 125

Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys

130 135 140

Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr

145 150 155 160

Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr

165 170 175

Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala

180 185 190

Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser

195 200 205

Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp

210 215 220

Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn Phe

225 230 235 240

Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala

245 250 255

Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser

260 265

<210> 11

<211> 1095

<212> DNA

＜213〉artificial sequence

<400> 11

atgaagccca ccctcatctc agtgcttgtg ataatattta tactcagagg aacaagagcc 60

cagagagtga ctcagcccga gaagctcctc tctgtcttta aaggggcccc agtggagctg 120

aagtgcaact attcctattc tgggagtcct gaactcttct ggtatgtcca gtactccaga 180

caacgcctcc agttactctt gagacacatc tctagagaga gcatcaaagg cttcactgct 240

gaccttaaca aaggcgagac atctttccac ctgaagaaac catttgctca agaggaagac 300

tcagccatgt attactgtgc tctaagtgcc tctgggggtt accagaaagt tacctttgga 360

actggaacaa agctccaagt catcccaaat atccagaacc ctgaccctgc cgtgtaccag 420

ctgagagact ctaaatccag tgacaagtct gtctgcctat tcaccgattt tgattctcaa 480

acaaatgtgt cacaaagtaa ggattctgat gtgtatatca cagacaaaac tgtgctagac 540

atgaggtcta tggacttcaa gagcaacagt gctgtggcct ggagcaacaa atctgacttt 600

gcatgtgcaa acgccttcaa caacagcatt attccagaag acaccttctt ccccagccca 660

gaaagttcct gtggggatct ggatcccaaa ctctgctacc tgctggatgg aatcctcttc 720

atctatggtg tcattctcac tgccttgttc ctgagagtga agttcagcag gagcgcagac 780

gcccccgcgt accagcaggg ccagaaccag ctctataacg agctcaatct aggacgaaga 840

gaggagtacg atgttttgga caagagacgt ggccgggacc ctgagatggg gggaaagccg 900

cagagaagga agaaccctca ggaaggcctg tacaatgaac tgcagaaaga taagatggcg 960

gaggcctaca gtgagattgg gatgaaaggc gagcgccgga ggggcaaggg gcacgatggc 1020

ctttaccagg gtctcagtac agccaccaag gacacctacg acgcccttca catgcaggcc 1080

ctaccccctc gctaa 1095

<210> 12

<211> 363

<212> PRT

＜213〉artificial sequence

<400> 12

Lys Pro Thr Leu Ile Ser Val Leu Val Ile Ile Phe Ile Leu Arg Gly

1 5 10 15

Thr Arg Ala Gln Arg Val Thr Gln Pro Glu Lys Leu Leu Ser Val Phe

20 25 30

Lys Gly Ala Pro Val Glu Leu Lys Cys Asn Tyr Ser Tyr Ser Gly Ser

35 40 45

Pro Glu Leu Phe Trp Tyr Val Gln Tyr Ser Arg Gln Arg Leu Gln Leu

50 55 60

Leu Leu Arg His Ile Ser Arg Glu Ser Ile Lys Gly Phe Thr Ala Asp

65 70 75 80

Leu Asn Lys Gly Glu Thr Ser Phe His Leu Lys Lys Pro Phe Ala Gln

85 90 95

Glu Glu Asp Ser Ala Met Tyr Tyr Cys Ala Leu Ser Ala Ser Gly Gly

100 105 110

Tyr Gln Lys Val Thr Phe Gly Thr Gly Thr Lys Leu Gln Val Ile Pro

115 120 125

Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys

130 135 140

Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr

145 150 155 160

Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr

165 170 175

Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala

180 185 190

Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser

195 200 205

Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Gly

210 215 220

Asp Leu Asp Pro Lys Leu Cys Tyr Leu Leu Asp Gly Ile Leu Phe Ile

225 230 235 240

Tyr Gly Val Ile Leu Thr Ala Leu Phe Leu Arg Val Lys Phe Ser Arg

245 250 255

Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn

260 265 270

Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg

275 280 285

Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn

290 295 300

Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu

305 310 315 320

Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly

325 330 335

His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr

340 345 350

Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

355 360

<210> 13

<211> 2391

<212> DNA

＜213〉artificial sequence

<400> 13

atggacacca gagtactttg ctgtgcggtc atctgtcttc tgggggcagg tctctcaaat 60

gccggcgtca tgcagaaccc aagacacctg gtcaggagga ggggacagga ggcaagactg 120

agatgcagcc caatgaaagg acacagtcat gtttactggt atcggcagct cccagaggaa 180

ggtctgaaat tcatggttta tctccagaaa gaaaatatca tagatgagtc aggaatgcca 240

aaggaacgat tttctgctga atttcccaaa gagggcccca gcatcctgag gatccagcag 300

gtagtgcgag gagattcggc agcttatttc tgtgccagct cacgcgcccg ggacggttcc 360

tacgagcagt acttcgggcc gggcaccagg ctcacggtca cagaggacct gaaaaacgtg 420

ttcccacccg aggtcgctgt gtttgagcca tcagaagcag agatctccca cacccaaaag 480

gccacactgg tgtgcctggc cacaggcttc taccccgacc acgtggagct gagctggtgg 540

gtgaatggga aggaggtgca cagtggggtc agcacagacc cgcagcccct caaggagcag 600

cccgccctca atgactccag atactgcctg agcagccgcc tgagggtctc ggccaccttc 660

tggcagaacc cccgcaacca cttccgctgt caagtccagt tctacgggct ctcggagaat 720

gacgagtgga cccaggatag ggccaaacct gtcacccaga tcgtcagcgc cgaggcctgg 780

ggtagagcag actgtgggga tctggatccc aaactctgct acctgctgga tggaatcctc 840

ttcatctatg gtgtcattct cactgccttg ttcctgagag tgaagttcag caggagcgca 900

gacgcccccg cgtaccagca gggccagaac cagctctata acgagctcaa tctaggacga 960

agagaggagt acgatgtttt ggacaagaga cgtggccggg accctgagat ggggggaaag 1020

ccgcagagaa ggaagaaccc tcaggaaggc ctgtacaatg aactgcagaa agataagatg 1080

gcggaggcct acagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa ggggcacgat 1140

ggcctttacc agggtctcag tacagccacc aaggacacct acgacgccct tcacatgcag 1200

gccctacccc ctcgcggctc cggagcaccg gtgaaacaga ctttgaattt tgaccttctc 1260

aagttggcgg gagacgtgga gtccaaccca gggcccatga agcccaccct catctcagtg 1320

cttgtgataa tatttatact cagaggaaca agagcccaga gagtgactca gcccgagaag 1380

ctcctctctg tctttaaagg ggccccagtg gagctgaagt gcaactattc ctattctggg 1440

agtcctgaac tcttctggta tgtccagtac tccagacaac gcctccagtt actcttgaga 1500

cacatctcta gagagagcat caaaggcttc actgctgacc ttaacaaagg cgagacatct 1560

ttccacctga agaaaccatt tgctcaagag gaagactcag ccatgtatta ctgtgctcta 1620

agtgcctctg ggggttacca gaaagttacc tttggaactg gaacaaagct ccaagtcatc 1680

ccaaatatcc agaaccctga ccctgccgtg taccagctga gagactctaa atccagtgac 1740

aagtctgtct gcctattcac cgattttgat tctcaaacaa atgtgtcaca aagtaaggat 1800

tctgatgtgt atatcacaga caaaactgtg ctagacatga ggtctatgga cttcaagagc 1860

aacagtgctg tggcctggag caacaaatct gactttgcat gtgcaaacgc cttcaacaac 1920

agcattattc cagaagacac cttcttcccc agcccagaaa gttcctgtgg ggatctggat 1980

cccaaactct gctacctgct ggatggaatc ctcttcatct atggtgtcat tctcactgcc 2040

ttgttcctga gagtgaagtt cagcaggagc gcagacgccc ccgcgtacca gcagggccag 2100

aaccagctct ataacgagct caatctagga cgaagagagg agtacgatgt tttggacaag 2160

agacgtggcc gggaccctga gatgggggga aagccgcaga gaaggaagaa ccctcaggaa 2220

ggcctgtaca atgaactgca gaaagataag atggcggagg cctacagtga gattgggatg 2280

aaaggcgagc gccggagggg caaggggcac gatggccttt accagggtct cagtacagcc 2340

accaaggaca cctacgacgc ccttcacatg caggccctac cccctcgcta a 2391

<210> 14

<211> 38

<212> DNA

＜213〉artificial sequence

<400> 14

ccgctcgagg ccaggatgga caccagagta ctttgctg 38

<210> 15

<211> 42

<212> DNA

＜213〉artificial sequence

<400> 15

agagtttggg atccagatcc ccacagtctg ctctacccca gg 42

<210> 16

<211> 45

<212> DNA

＜213〉artificial sequence

<400> 16

ggtagagcag actgtgggga tctggatccc aaactctgct acctg 45

<210> 17

<211> 46

<212> DNA

＜213〉artificial sequence

<400> 17

gtttcaccgg tgctccggag ccgcgagggg gtagggcctg catgtg 46

<210> 18

<211> 97

<212> DNA

＜213〉artificial sequence

<400> 18

gcaccggtga aacagacttt gaattttgac cttctcaagt tggcgggaga cgtggagtcc 60

aacccagggc ccatgaagcc caccctcatc tcagtgc 97

<210> 19

<211> 42

<212> DNA

＜213〉artificial sequence

<400> 19

agagtttggg atccagatcc ccacaggaac tttctgggct gg 42

<210> 20

<211> 43

<212> DNA

＜213〉artificial sequence

<400> 20

cccagaaagt tcctgtgggg atctggatcc caaactctgc tac 43

<210> 21

<211> 42

<212> DNA

＜213〉artificial sequence

<400> 21

ataagaatgc ggccgcttag cgagggggta gggcctgcat gt 42

Claims (10)

1.HIV-1 peptide Env _120-128Specific t-cell receptor (TCR) comprises α chain and β chain, and wherein, the described sequence of SEQ ID NO:3 is contained in the CDR3 district of α chain; The sequence shown in the SEQ ID NO:4 is contained in the CDR3 district of β chain.

2. HIV-1 peptide Env according to claim 1 _120-128Specificity TCR, it is characterized in that, the α chain of described TCR is to be substituted, to lack and/or increased the aminoacid sequence that one or more amino acid and/or the special and exogenous β chain of end modified rear resulting energy are assembled into the TCR protein molecular by the aminoacid sequence shown in the SEQ ID NO:10; The β chain is to be substituted, to lack and/or increased the aminoacid sequence that one or more amino acid and/or the special and exogenous α chain of end modified rear resulting energy are assembled into the TCR protein molecular by the aminoacid sequence shown in the SEQ ID NO:6.

3. HIV-1 peptide Env according to claim 2 _120-128Specificity TCR is characterized in that, the aminoacid sequence of described α chain is shown in SEQ ID NO:12, and the aminoacid sequence of β chain is shown in SEQ ID NO:8.

4. the gene of coding claim 1～3 each described TCR.

5. HIV-1 peptide Env _120-128The fusion gene of specificity TCR, its sequence is shown in SEQ ID NO:13.

6. a recombinant retroviral vector contains claim 4 or 5 described genes.

7. recombinant retroviral vector according to claim 6 is characterized in that, the carrier that sets out comprises pMX-IRES-GFP, pMCs-IRES-GFP or pMYx-IRES-GFP.

8. claim 6 or the 7 described recombinant retroviral vectors retrovirus that obtains after packing.

9. the CD8 of Retroviral Transfer claimed in claim 8 ⁺The T cell.

10. each described HIV-1 peptide Env of claim 1～3 _120-128The CD8 of specificity TCR, claim 4 or 5 described genes, claim 6 or 7 described recombinant retroviral vectors, retrovirus claimed in claim 8, Retroviral Transfer claimed in claim 9 ⁺The T cell is at the preparation anti-AIDS ,Application in AIDS/tuberculosis coinfection disease medicament.

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